This renewal application is a continuation of efforts to detect colorless biomolecules in the visible spectral region by designing synthetic organic indicators with high analyte specificity. The broad, long-term objective of this program is to facilitate the diagnosis and understanding of disease via the creation of practical new tools for biomedical research. Elevated plasma homocysteine (Hcy) levels are associated with increased risk of myocardial infarction, stroke and venous thromboembolism. Homocysteine has also been linked to increased risk of Alzheimer's disease, neural tube defects and complications during pregnancy, inflammatory bowel disease and osteoporosis. Despite over 20 years of intense study, the actual role of homocysteine in disease remains unclear. The goals are to contribute to (i) clarifying the current understanding of the role of homocysteine in disease and (ii) facilitate the study homocysteine in natural media. The approach to these two issues is rooted in fundamental organic chemistry. Uncovering the differences in the basic organic chemistry between Hcy and other thiols and biomolecules will afford new knowledge relevant to Hcy's involvement in disease and concurrently lead to highly selective and enabling new detection methods. To approach these goals, the following Specific Aims are proposed: Aim 1. Highly selective indicators for simplifying the determination of homocysteine. Aim 2. New insights into the chemistry of the homocysteine-derived alpha-amino carbon-centered radical. Aim 3. Detection and chemistry of protein-bound homocystamide. Each of the Specific Aims of this project involves improving the detection of homocysteine. The presence of homocysteine above certain levels in humans often indicates serious risk for disease. This useful amino acid biomarker has been studied for decades, but its role in disease is still not clear. We propose to create simpler new methods and materials for homocysteine detection as well as clarification of its role in disease.